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PubMed1995

Processivity of Escherichia coli and rat liver mitochondrial uracil-DNA glycosylase is affected by NaCl concentration.

Escherichia coli uracil-DNA glycosylase was shown to catalyze the hydrolysis of a site-specific uracil residue from a defined single-stranded oligonucleotide (25-mer). With duplex 25-mer, the rate of uracil removal from double-stranded DNA containing a U.G mispair was approximately 2-fold greater than a U.A base pair. The mechanism by which E. coli and rat liver mitochondrial uracil-DNA glycosylase located sequential uracil residues within double-stranded DNA was investigated. Two concatemeric polynucleotide substrates were constructed by ligation of homologous 5'-end 32P-labeled 25-mer double-stranded oligonucleotides that contained either a site-specific U.G or U.A target site at intervals of 25 nucleotides along one strand of the DNA. Reaction of uracil-DNA glycosylase with these concatemeric DNAs, followed by alkaline hydrolysis of the resultant AP-sites, would produce predominantly [32P]25-mer products, if a processive mechanism was used to locate successive uracil residues, or oligomeric multiples of [32P]25-mer, if a distributive mode was exhibited. Both the bacterial and the mitochondrial enzymes were found to act processively on U.A- and U.G-containing DNA in the absence of NaCl, based on the initial rate of 25-mer produced relative to the total amount of uracil excised. Approximately 50% of the total uracil excised resulted in the release of 25-mer product. The addition of NaCl (> or = 50 mM) caused reduced processivity on both U.A- and U.G-containing DNA substrates. The mode of action of uracil-DNA glycosylase was very similar to that observed for the EcoRI endonuclease cleavage of restriction sites contained in the same DNA substrate which was used as a positive control.

Animals↗

PubMed1997

Contrasting effects of single stranded DNA binding protein on the activity of uracil DNA glycosylase from Escherichia coli towards different DNA substrates.

Excision of uracil from tetraloop hairpins and single stranded ('unstructured') oligodeoxyribonucleotides by Escherichia coli uracil DNA glycosylase has been investigated. We show that, compared with a single stranded reference substrate, uracil from the first, second, third and the fourth positions of the loops is excised with highly variable efficiencies of 3.21, 0.37, 5.9 and 66.8%, respectively. More importantly, inclusion of E.coli single stranded DNA binding protein (SSB) in the reactions resulted in approximately 7-140-fold increase in the efficiency of uracil excision from the first, second or the third position in the loop but showed no significant effect on its excision from the fourth position. In contrast, the presence of SSB decreased uracil excision from the single stranded ('unstructured') substrates approximately 2-3-fold. The kinetic studies show that the increased efficiency of uracil release from the first, second and the third positions of the tetraloops is due to a combination of both the improved substrate binding and a large increase in the catalytic rates. On the other hand, the decreased efficiency of uracil release from the single stranded substrates ('unstructured') is mostly due to the lowering of the catalytic rates. Chemical probing with KMnO4showed that the presence of SSB resulted in the reduction of cleavage of the nucleotides in the vicinity of dUMP residue in single stranded substrates but their increased susceptibility in the hairpin substrates. We discuss these results to propose that excision of uracil from DNA-SSB complexes by uracil DNA glycosylase involves base flipping. The use of SSB in the various applications of uracil DNA glycosylase is also discussed.

Base Sequence↗

PubMed2005

Physiologically based pharmacokinetic modelling of the three-step metabolism of pyrimidine using C-uracil as an in vivo probe.

AIMS: Approximately 80% of uracil is excreted as beta-alanine, ammonia and CO2 via three sequential reactions. The activity of the first enzyme in this scheme, dihydropyrimidine dehydrogenase (DPD), is reported to be the key determinant of the cytotoxicity and side-effects of 5-fluorouracil. The aim of the present study was to re-evaluate the pharmacokinetics of uracil and its metabolites using a sensitive assay and based on a newly developed, physiologically based pharmacokinetic (PBPK) model. METHODS: [2-(13)C]Uracil was orally administrated to 12 healthy males at escalating doses of 50, 100 and 200 mg, and the concentrations of [2-(13)C]uracil, [2-(13)C]5,6-dihydrouracil and beta-ureidopropionic acid (ureido-(13)C) in plasma and urine and (13)CO2 in breath were measured by liquid chromatography-tandem mass spectrometry and gas chromatograph-isotope ratio mass spectrometry, respectively. RESULTS: The pharmacokinetics of [2-(13)C]uracil were nonlinear. The elimination half-life of [2-(13)C]5,6-dihydrouracil was 0.9-1.4 h, whereas that of [2-(13)C]uracil was 0.2-0.3 h. The AUC of [2-(13)C]5,6-dihydrouracil was 1.9-3.1 times greater than that of [2-(13)C]uracil, whereas that of ureido-(13)C was 0.13-0.23 times smaller. The pharmacokinetics of (13)CO2 in expired air were linear and the recovery of (13)CO2 was approximately 80% of the dose. The renal clearance of [2-(13)C]uracil was negligible. CONCLUSION: A PBPK model to describe (13)CO2 exhalation after orally administered [2-(13)C]uracil was successfully developed. Using [2-(13)C]uracil as a probe, this model could be useful in identifying DPD-deficient patients at risk of 5-fluorouracil toxicity.

Adult↗

PubMed1981

Recombination of uracil-containing lambda bacteriophages.

Controlled incorporation of uracil into the deoxyribonucleic acid (DNA) of lambda bacteriophages was achieved by growth on dut ung thy mutants of Escherichia coli. The frequency of substitution of uracil for thymine, estimated by alkaline sucrose sedimentation of phage DNA treated in vitro with uracil DNA glycosylase, ranged from 0.17 to 1.9%. The corresponding ratio between the plating efficiencies on wild-type (Ung+) and glycosylase-deficient (Ung-) bacteria ranged from 0.70 to 0.05. If a single-hit dependence of plating efficiency on uracil content is assumed, the probability that any given uracil residue is lethal is approximately 1% (about one-fifth the probability for a pyrimidine dimer). The effect of uracil on recombination was studied in experiments with lambda tandem duplication phages (ethylenediaminetetraacetic acid [EDTA] sensitive), which are converted to single-copy phages (EDTA resistant) by general recombination. For repressed infections (of homoimmune lysogens), recombination was measured by a two-stage assay (DNA extraction, transfection of spheroplasts, and EDTA treatment). The frequencies observed for uracil-containing phages (2 to 4%) were 5 to 10 times higher than control values. However, comparisons with ultraviolet irradiated phages indicated that uracil residues promoted recombination less than 1/100 as efficiently as ultraviolet-induced lesions. Recombination of uracil-containing phages during repressed infections was negligible in recA and partially reduced in recB bacteria. Recombination was very low in ung cells, suggesting that excision repair was responsible for the stimulation. Interestingly, uracil-stimulated recombination was elevated about twofold in xth bacteria.

Bacteriophage lambda↗

PubMed1985

Isolation of insertion, deletion, and nonsense mutations of the uracil-DNA glycosylase (ung) gene of Escherichia coli K-12.

Two uracil-DNA glycosylase (ung) mutation selection procedures based upon the ability of uracil glycosylase to degrade the chromosomes of organisms containing uracil-DNA were devised to obtain a collection of well-defined ung alleles. In an enrichment procedure, lysogens were selected from Escherichia coli cultures infected with lambda pKanr phage containing uracil in their DNA. (These uracil-DNA phage were prepared by growth on host cells deficient in both dUTPase and uracil-DNA glycosylase.) The lysogenic Kanr population was enriched for uracil glycosylase-deficient mutants by a factor of 10(4). In a phage suicide selection procedure, lambda pung+ phage were unable to form plaques on dut ung cells containing uracil-DNA in their chromosomes, and all of the progeny were lambda pung-. Deletion, insertion (ung::Mu and ung::Tn10), nonsense, and missense mutants were isolated by using these procedures. Extracts of three insertion mutants contained no detectable enzyme activity. All of the other mutant isolates had less than 1% of the normal uracil glycosylase specific activity. The previously studied ung-1 allele, which was derived by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis, produced about 0.02% of the normal amount of uracil glycosylase activity. No significant phenotypic differences between ung-1 and ung::Tn10 alleles were observed. Variations of the lysogen selection procedure may be helpful for isolating other DNA glycosylase mutations in E. coli and other organisms.

Alleles↗

PubMed1992

Carcinogenicity of uracil, a nongenotoxic chemical, in rats and mice and its rationale.

In Experiment 1, groups of thirty 6-wk-old male and female F344 rats were given diets containing 0 (control) or 3% uracil for 104 wk. In the uracil-treated groups, carcinomas, in particular transitional cell carcinomas, developed in the urinary bladder of 90% of the males and 19% of the females. Squamous cell carcinomas also developed in 10% of the males, but not in females. Striking findings were that calculi were present in the urinary bladder of almost all males, but in only 30% of the females, and that induction of urinary bladder carcinomas was related to the presence of calculi. In Experiment 2, groups of thirty 6-wk-old male and female C57BL/6 x C3H F1 mice were given a diet containing 0 (control) or 3% (from Wk 1 to Wk 6) and 2.5% (from Wk 7 to Wk 96) uracil. The total observation period was 96 wk. In the uracil-treated groups, transitional cell carcinomas developed in 76% of the females and 8% of the males. Squamous cell carcinomas developed in only 8% of the males. In Experiment 3, 6-wk-old male F344 rats were given diets containing 3% uracil, 3% uracil plus 5% or 10% NaCl, or 10% NaCl for 36 wk and then diet without chemicals for a recovery period of 4 wk. The incidences of carcinomas and calculi of the urinary bladder were 75% and 81% in the group given uracil alone, 6% and 6% in the group given uracil plus 5% NaCl, and both zero in the group given uracil plus 10% NaCl. Thus, the present study showed that the inductions of urinary bladder carcinomas by uracil, a nongenotoxic compound, in rats and mice showed sex differences and were related to the presence of calculi in the urinary bladder.

Animals↗

PubMed1986

Uracil nucleotide synthesis in a human breast cancer cell line (MCF-7) and in two drug-resistant sublines that contain increased levels of enzymes of the de novo pyrimidine pathway.

Cultured wild-type MCF-7 human breast cancer cells and two MCF-7 sublines that overproduce enzymes of the de novo pyrimidine biosynthetic pathway were compared with regard to: rate of de novo biosynthesis of uracil nucleotides, sensitivity of the de novo and salvage pathways to the concentration of intracellular uracil nucleotides, and potential of exogenous uridine at concentrations equivalent to plasma levels to affect de novo pyrimidine biosynthesis. The PALAR MCF-7 subline, which is resistant to N-(phosphonacetyl)-L-aspartate and has 5.2 times the activity of the first de novo enzyme as the wild-type MCF-7 cells, synthesizes uracil nucleotides via the de novo pathway at a rate that is 5.8 times that of the wild type MCF-7 cells. The PYRR MCF-7 subline, which is resistant to pyrazofurin and has 15.1 times the activity of orotate phosphoribosyltransferase as the wild-type MCF-7 cells, synthesizes uracil nucleotides via the de novo pathway at a rate that is 1.4 times that of wild-type MCF-7 cells. These results are consistent with carbamyl phosphate synthetase being the rate-controlling step of de novo pyrimidine biosynthesis. In the presence of exogenous uridine at concentrations equivalent to that found in plasma (4.4-8.6 microM), the uracil nucleotide pool of wild-type MCF-7 cells was expanded by 20% and de novo synthesis was inhibited by 55%. Incubation of PALAR MCF-7 cells with uridine at concentrations between 7.3 and 16.8 microM caused a 40% increase in the uracil nucleotide pool and a 30% inhibition of de novo synthesis. De novo synthesis of uracil nucleotides in PYRR MCF-7 cells was not affected by a greater than 10-fold increase in the uracil nucleotide pool. Salvage of [14C] uridine was inhibited by an expanded uracil nucleotide pool in the wild-type and PYRR MCF-7 cells but was not inhibited in the PALAR MCF-7 cell line. These results demonstrate that, although the overproduced enzymes exhibit substrate affinities and specificities in cell-free preparations similar to those of the wild-type enzymes, in intact cells the resistant cell lines exhibit marked differences in the control of de novo and salvage pyrimidine biosynthetic pathways by intracellular uracil nucleotides.

Amides↗

PubMed1987

Strong promoting activity of reversible uracil-induced urolithiasis on urinary bladder carcinogenesis in rats initiated with N-butyl-N-(4-hydroxybutyl)nitrosamine.

The tumor-promoting effect of uracil-induced calculi on rat urinary bladder carcinogenesis was investigated in male F344 rats pretreated with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). Since uracil-induced calculi and papillomatosis of the bladder are reversible, uracil was given for a limited period after the treatment with BBN. Animals were given 0.05% BBN in their drinking water for 4 wk and then treated with uracil as 3% of the diet for 8 or 16 wk. After the uracil treatment, rats were given basal diet without uracil until Wk 28 of the experiment. Animals were killed from each group at the end of either Wk 12, 20, or 28. The incidence of carcinoma of the bladder was 40% after only 8 wk of uracil treatment following BBN initiation and increased to 100% when uracil treatment was extended to 16 wk. After discontinuation of uracil treatment, the papillomatosis disappeared, but the incidence of carcinoma steadily increased with increasing time. In the control group given BBN alone, only 1 of 16 rats had carcinoma at Wk 28. The present findings clearly demonstrate that uracil-induced urolithiasis had a strong promoting activity on BBN bladder carcinogenesis.

Animals↗

PubMed1983

Partial purification and characterization of a uracil-DNA glycosylase from wheat germ.

A uracil-DNA glycosylase has been purified over 1,000-fold from wheat germ, the first such repair activity isolated from a higher plant. The enzyme has a molecular weight of approximately 27,000 and is resistant to metal ion chelators, but inhibited by high concentrations of either mono or divalent cations. This glycosylase is unable to release uracil from the mononucleotides dUMP and dUTP or from wheat germ RNA. Twelve pyrimidine analogues which closely mimic uracil structurally and the nucleoside uridine were examined for their ability to inhibit glycosylase activity. However, only 5-azauracil and 6-aminouracil inhibited enzymatic release of uracil to the same degree as uracil itself. An inhibitor induced by bacteriophage T5 which inhibits Escherichia coli uracil-DNA glycosylase has been shown not to affect the glycosylase isolated from wheat germ, indicating that these two enzymes differ. The ability of the wheat germ uracil-DNA glycosylase to completely remove available uracil from synthetic DNA substrates in which thymine had been replaced by uracil in varying percentages was also examined and found not to depend on percentage of uracil in the substrates.

DNA Glycosylases↗

PubMed1986

Purification and some properties of uracil phosphoribosyltransferase from Escherichia coli K12.

Uracil phosphoribosyltransferase from Escherichia coli K12 was purified to homogeneity as determined by polyacrylamide gel electrophoresis. For this purpose a pyrimidine-requiring strain harboring the upp gene on a ColE1 plasmid was used, which showed 15-times higher uracil phosphoribosyltransferase activity in a crude extract. When this strain was grown under conditions of uracil starvation, an additional 10-times elevation of the enzyme activity was obtained. The molecular weight of uracil phosphoribosyltransferase was determined to be 75000; the enzyme consists of three subunits with a molecular weight of 23500. Uracil phosphoribosyltransferase is specific for uracil and some uracil analogues. The apparent Km values for uracil and PRib-PP were 7 microM and 300 microM, respectively. As an effector of enzyme activity, GTP lowered the Km for PRib-PP to 90 microM and increased the Vmax value 2-fold, but had no effect on the Km for uracil. The effect of GTP was found to be pH-dependent. The enzymatic characterization of uracil phosphoribosyltransferase and the observed regulation of its synthesis emphasizes the role of the enzyme in pyrimidine salvage.

Chromatography, Affinity↗

PubMed2001

Uracil-initiated base excision DNA repair synthesis fidelity in human colon adenocarcinoma LoVo and Escherichia coli cell extracts.

The error frequency of uracil-initiated base excision repair (BER) DNA synthesis in human and Escherichia coli cell-free extracts was determined by an M13mp2 lacZ alpha DNA-based reversion assay. Heteroduplex M13mp2 DNA was constructed that contained a site-specific uracil target located opposite the first nucleotide position of opal codon 14 in the lacZ alpha gene. Human glioblastoma U251 and colon adenocarcinoma LoVo whole-cell extracts repaired the uracil residue to produce form I DNA that was resistant to subsequent in vitro cleavage by E. coli uracil-DNA glycosylase (Ung) and endonuclease IV, indicating that complete uracil-initiated BER repair had occurred. Characterization of the BER reactions revealed that (1) the majority of uracil-DNA repair was initiated by a uracil-DNA glycosylase-sensitive to Ugi (uracil-DNA glycosylase inhibitor protein), (2) the addition of aphidicolin did not significantly inhibit BER DNA synthesis, and (3) the BER patch size ranged from 1 to 8 nucleotides. The misincorporation frequency of BER DNA synthesis at the target site was 5.2 x 10(-4) in U251 extracts and 5.4 x 10(-4) in LoVo extracts. The most frequent base substitution errors in the U251 and LoVo mutational spectrum were T to G > T to A >> T to C. Uracil-initiated BER DNA synthesis in extracts of E. coli BH156 (ung) BH157 (dug), and BH158 (ung, dug) was also examined. Efficient BER occurred in extracts of the BH157 strain with a misincorporation frequency of 5.6 x 10(-4). A reduced, but detectable level of BER was observed in extracts of E. coli BH156 cells; however, the mutation frequency of BER DNA synthesis was elevated 6.4-fold.

Adenocarcinoma↗

PubMed2002

Presteady-state analysis of a single catalytic turnover by Escherichia coli uracil-DNA glycosylase reveals a "pinch-pull-push" mechanism.

Uracil-DNA glycosylase catalyzes the excision of uracils from DNA via a mechanism where the uracil is extrahelically flipped out of the DNA helix into the enzyme active site. A conserved leucine is inserted into the DNA duplex space vacated by the uracil leading to the paradigmatic "push-pull" mechanism of nucleotide flipping. However, the order of these two steps during catalysis has not been conclusively established. We report a complete kinetic analysis of a single catalytic turnover using a hydrolyzable duplex oligodeoxyribonucleotide substrate containing a uracil:2-aminopurine base pair. Rapid chemical-quenched-flow methods defined the kinetics of excision at the active site during catalysis. Stopped-flow fluorometry monitoring the 2-aminopurine fluorescence defined the kinetics of uracil flipping. Parallel experiments detecting the protein fluorescence showed a slower Leu(191) insertion step occurring after nucleotide flipping but before excision. The inserted Leu(191) acts as a doorstop to prevent the return of the flipped-out uracil residue, thereby facilitating the capture of the uracil in the active site and does not play a direct role in "pushing" the uracil out of the DNA helix. The results define for the first time the proper sequence of events during a catalytic cycle and establish a "pull-push", as opposed to a "push-pull", mechanism for nucleotide flipping.

Binding Sites↗

PubMed1999

Differential effects of single-stranded DNA binding proteins (SSBs) on uracil DNA glycosylases (UDGs) from Escherichia coli and mycobacteria.

Deamination of cytosines results in accumulation of uracil residues in DNA, which unless repaired lead to GC-->AT transition mutations. Uracil DNA glyco-sylase excises uracil residues from DNA and initiates the base excision repair pathway to safeguard the genomic integrity. In this study, we have investigated the effect of single-stranded DNA binding proteins (SSBs) from Escherichia coli (Eco SSB) and Mycobacterium tuberculosis (Mtu SSB) on uracil excision from synthetic substrates by uracil DNA glycosylases (UDGs) from E. coli, Mycobacterium smegmatis and M.tuberculosis (referred to as Eco -, Msm - and Mtu UDGs respectively). Presence of SSBs with all the three UDGs resulted in decreased efficiency of uracil excision from a single-stranded 'unstructured' oligonucleo-tide, SS-U9. On the other hand, addition of Eco SSB to Eco UDG, or Mtu SSB to Mtu UDG reactions resulted in increased efficiency of uracil excision from a hairpin oligonucleotide containing dU at the second position in a tetraloop (Loop-U2). Interestingly, the efficiency of uracil excision by Msm UDG from the same substrate was decreased in the presence of either Eco- or Mtu SSBs. Furthermore, Mtu SSB also decreased uracil excision from Loop-U2 by Eco UDG. Our studies using surface plasmon resonance technique demonstrated interactions between the homologous combinations of SSBs and UDGs. Heterologous combinations either did not show detectable interaction (Eco SSB with Mtu UDG) or showed a relatively weaker interaction (Mtu SSB with Eco UDG). Taken together, our studies suggest differential interactions between the two groups (SSBs and UDGs) of the highly conserved proteins. Such studies may provide important clues to design selective inhibitors against this important class of DNA repair enzymes.

DNA↗

PubMed2002

dUTPase and uracil-DNA glycosylase are central modulators of antifolate toxicity in Saccharomyces cerevisiae.

The thymidylate synthase reaction remains an important target for widely used anticancer agents; however, the clinical utility of these drugs is limited by the occurrence of cellular resistance. Despite the considerable amount of information available regarding mechanisms of drug action, the relative significance of downstream events that result in lethality remains unclear. In this study, we have developed a model system using the budding yeast Saccharomyces cerevisiae to dissect the influence of dUMP misincorporation into DNA as a contributing mechanism of cytotoxicity induced by antifolate agents. The activities of dUTPase and uracil-DNA glycosylase, key enzymes in uracil-DNA metabolism, were diminished or augmented, and the manipulated strains were analyzed for biochemical endpoints of toxicity. Cells overexpressing dUTPase were protected from cytotoxicity by their ability to prevent dUTP pool expansion and were able to recover from an early S-phase checkpoint arrest. In contrast, depletion of dUTPase activity leads to the accumulation of dUTP pools and enhanced sensitivity to antifolates. These cells were also arrested in early S-phase and were unable to complete DNA replication after drug withdrawal, resulting in lethality. Inactivation of uracil base excision repair induced partial resistance to early cytotoxicity (within 10 h); however, lethality ultimately resulted at later time points (12-24 h), presumably because of the detrimental effects of stable uracil misincorporation. Although these cells were able to complete replication with uracil-substituted DNA, they arrested at the G(2)-M phase. This finding may represent a novel mechanism by which the G(2)-M checkpoint is signaled by the presence of uracil-substituted DNA. Together these data provide both genetic and biochemical evidence demonstrating that lethality from antifolates in yeast is primarily dependent on uracil misincorporation into DNA, and that uracil-independent mechanisms associated with dTTP depletion play a minor role. Our findings indicate that the relative expression levels of both dUTPase and uracil-DNA glycosylase can have great influence over the efficacy of thymidylate synthase-directed chemotherapy, thereby enhancing the candidacy of these proteins as prognostic markers and alternative targets for therapeutic development.

DNA Damage↗

PubMed2001

Uracil salvage pathway in PC12 cells.

The salvage anabolism of uracil to pyrimidine ribonucleosides and ribonucleotides was investigated in PC12 cells. Pyrimidine base phosphoribosyl transferase is absent in PC12 cells. As a consequence any uracil or cytosine salvage must be a 5-phosphoribosyl 1-pyrophosphate-independent process. When PC12 cell extracts were incubated with ribose 1-phosphate, ATP and uracil they can readily catalyze the synthesis of uracil nucleotides, through a salvage pathway in which the ribose moiety of ribose 1-phosphate is transferred to uracil via uridine phosphorylase (acting anabolically), with subsequent uridine phosphorylation. This pathway is similar to that previously described by us in rat liver and brain extracts (Cappiello et al., Biochim. Biophys. Acta 1425 (1998) 273; Mascia et al., Biochim. Biophys. Acta 1472 (1999) 93). We show using intact PC12 cells that they can readily take up uracil from the external medium. The analysis of intracellular metabolites reveals that uracil taken up is salvaged into uracil nucleotides, with uridine as an intermediate. We propose that the ribose 1-phosphate-dependent uracil salvage shown by our in vitro studies, using tissues or cellular extracts, might also be operative in intact cells. Our results must be taken into consideration for the comprehension of novel chemotherapeutics' influence on pyrimidine neuronal metabolism.

Animals↗

PubMed1999

Specificity and catalysis of uracil DNA glycosylase. A molecular dynamics study of reactant and product complexes with DNA.

The structure of uracil DNA glycosylase (UDG) in complex with a nonamer duplex DNA containing a uracil has been determined only in the product state. The reactant state was constructed by reattaching uracil to the deoxyribose, and both complexes were studied by molecular dynamics simulations. Significant changes in the positions of secondary structural elements in the enzyme are induced by the hydrolysis of the glycosidic bond. The simulations show that the specificity of the uracil pocket in the enzyme is largely retained in both complexes with the exception of Asn-204, which has been identified as a residue that contributes to discrimination between uracil and cytosine. The hydrogen bond between the amide group of Asn-204 and O(4) of uracil is disrupted by fluctuations of the side chain in the reactant state and is replaced by a hydrogen bond to water molecules trapped in the interior of the protein behind the uracil binding pocket. The role of two residues implicated by mutation experiments to be important in catalysis, His-268 and Asp-145, is clarified by the simulations. In the reactant state, His-268 is found 3.45 +/- 0.34 A from the uracil, allowing a water molecule to form a bridge to O(2). The environment in the enzyme raises the pK(a) value of His-268 to 7.1, establishing a protonated residue for assisting in the hydrolysis of the glycosidic bond. In agreement with the crystallographic structure, the DNA backbone retracts after the hydrolysis to allow His-268 to approach the O(2) of uracil with a concomitant release of the bridging water molecule and a reduction in the pK(a) to 5.5, which releases the proton to the product. The side chain of Asp-145 is fully solvated in the reactant state and H-bonded through a water molecule to the 3'-phosphate of uridine. Both the proximity of Asp-145 to the negatively charged phosphate and its pK(a) of 4.4 indicate that it cannot act as a general base catalyst. We propose a mechanism in which the bridging water between Asp-145 and the 3'-phosphate accepts a proton from another water to stabilize the bridge through a hydronium ion as well as to produce the hydroxide anion required for the hydrolytic step. The mechanism is consistent with known experimental data.

Aspartic Acid↗

PubMed2006

Proton transfers induced by lead(II) in a uracil nucleobase: a study based on quantum chemistry calculations.

Within the context of metal biotoxicity, electrospray ionization mass spectrometry experiments (ESIMS) have recently been performed by us on the pyrimidine nucleobases (B) uracil and thymine complexed with lead(II) [Int. J. Mass. Spectrom. 2005, 243, 279]. Among the ions detected, [Pb(B)-H]+ complexes, where the base has been deprotonated, have been identified as producing intense signals. In the same study, quantum calculations based on density functional theory (DFT) have assessed the complexation sites and energies of [Pb(B)-H]+ ions. The present DFT investigations aim at giving an understanding on the energetics and mechanisms associated with uracil's loss of a proton. We specifically assess and quantify the role of lead binding in this process. For that purpose, intra- and intermolecular proton transfers have been considered. We have found that uracil (U) 1,3-tautomerization can be exergonic when uracil is complexed with Pb2+, in opposition to the situation without lead. The corresponding intramolecular processes were nonetheless found to occur at geological time scales. In contrast, the addition of a second body to [Pb(U)]2+ complexes, namely OH- or H2O (as found in the initial water droplet of ESIMS experiments), gives exergonic and fast uracil 1,3-proton transfers. Finally, we have shown that intermolecular proton transfers in uracil-H2O, uracil-OH-, or uracil-uracil complexes are able to explain the experimentally detected [Pb(U)-H]+ ions.

Cations, Divalent↗

PubMed2000

Fidelity of uracil-initiated base excision DNA repair in Escherichia coli cell extracts.

The error frequency and mutational specificity associated with Escherichia coli uracil-initiated base excision repair were measured using an M13mp2 lacZalpha DNA-based reversion assay. Repair was detected in cell-free extracts utilizing a form I DNA substrate containing a site-specific uracil residue. The rate and extent of complete uracil-DNA repair were measured using uracil-DNA glycosylase (Ung)- or double-strand uracil-DNA glycosylase (Dug)-proficient and -deficient isogenic E. coli cells. In reactions utilizing E. coli NR8051 (ung(+) dug(+)), approximately 80% of the uracil-DNA was repaired, whereas about 20% repair was observed using NR8052 (ung(-) dug(+)) cells. The Ung-deficient reaction was insensitive to inhibition by the PBS2 uracil-DNA glycosylase inhibitor protein, implying the involvement of Dug activity. Under both conditions, repaired form I DNA accumulated in conjunction with limited DNA synthesis associated with a repair patch size of 1-20 nucleotides. Reactions conducted with E. coli BH156 (ung(-) dug(+)), BH157 (ung(+) dug(-)), and BH158 (ung(-) dug(-)) cells provided direct evidence for the involvement of Dug in uracil-DNA repair. The rate of repair was 5-fold greater in the Ung-proficient than in the Ung-deficient reactions, while repair was not detected in reactions deficient in both Ung and Dug. The base substitution reversion frequency associated with uracil-DNA repair was determined to be approximately 5.5 x 10(-)(4) with transversion mutations dominating the mutational spectrum. In the presence of Dug, inactivation of Ung resulted in up to a 7.3-fold increase in mutation frequency without a dramatic change in mutational specificity.

Base Sequence↗